1. Field of the Invention
The present invention relates to a novel enzyme involved in the control of plant growth, DNA sequences coding for the enzyme and uses of the nucleotide sequence coding for the enzyme in the production of transgenic plants with improved or altered growth characteristics.
2. Related Art
The present invention relates to a novel enzyme involved in the control of plant growth, DNA sequences coding for the enzyme and uses of the nucleotide sequence coding for the enzyme in the production of transgenic plants with improved or altered growth characteristics.
The gibberellins (GAs) are a large group of diterpenoid carboxylic acids that are present in all higher plants and some fungi. Certain members of the group function as plant hormones and are involved in many developmental processes, including seed germination, stem extension, leaf expansion, flower initiation and development, and growth of the seeds and fruit. The biologically active GAs are usually C19 compounds containing a 19-10 lactone, a C-7 carboxylic acid and a 3β-hydroxyl group. The later stages of their biosynthesis involve the oxidative removal of C-20 and hydroxylation at C-3. Hydroxylation at the 2β position results in the production of biologically inactive products. This reaction is the most important route for GA metabolism in plants and ensures that the active hormones do not accumulate in plant tissues. The GA biosynthetic enzymes 7-oxidase, 20-oxidase, 3β-hydroxylase and 2β-hydroxylase are all 2-oxoglutarate dependent dioxygenases. These are a large group of enzymes for which 2-oxoglutarate is a co-substrate that is decarboxylated to succinate as part of the reaction (see review by Hedden, P. and Kamiya, Y., in Annu. Rev. Plant Physiol. Plant Mol. Biol. 48 431460 (1997)).
Chemical regulators of plant growth have been used in horticulture and agriculture for many years. Many of these compounds function by changing the GA concentration in plant tissues. For example, growth retardants inhibit the activity of enzymes involved in GA biosynthesis and thereby reduce the GA content. Such chemicals are used commonly, for example, to prevent lodging in cereals and to control the growth of ornamental and horticultural plants. Conversely, GAs may be applied to plants, such as in the application of GA3 to seedless grapes to improve the size and shape of the berry, and to barley grain to improve malt production. Mixtures of GA4 and GA7 are applied to apples to improve fruit quality and to certain conifers to stimulate cone production. There are several problems associated with the use of growth regulators. Some of the growth retardants are highly persistent in the soil making it difficult to grow other crops following a treated crop. Others require repeated applications to maintain the required effect. It is difficult to restrict application to the target plant organs without it spreading to other organs or plants and having undesirable effects. Precise targeting of the growth-regulator application can be very labour intensive. A non-chemical option for controlling plant morphology is, thus, highly desirable.
Developing seeds often contain high concentrations of GAs and relatively large amounts of GA-biosynthetic enzymes. Mature seeds of runner bean (Phaseolus coccineus) contain extremely high concentrations of the 2β-hydroxy GA, GA8, as its glucoside, indicating that high levels of 2β-hydroxylase activity must be present. This has been confirmed for the related species Phaseolus vulgaris in which there is a rapid increase in GA 2β-hydroxylase activity shortly before seeds reach full maturity (Albone et al., Planta 177 108-115 (1989)). 2β-Hydroxylases have been partially purified from the cotyledons of Pisum sativum (Smith, V. A. and MacMillan, J., Planta 167 9-18 (1983)) and Phaseolus vulgaris (Griggs et al Phytochemistry 30 2507-2512 (1991) and Smith, V. A. and MacMillan, J., J. Plant Growth Regul. 2 251-264 (1984)). These studies showed that there was evidence that, for both sources, at least two enzymes with different substrate specificities are present. Two activities from cotyledons of imbibed P. vulgaris were separable by cation-exchange chromatography and gel-filtration. The major activity, corresponding to an enzyme of Mr 26,000 by size exclusion HPLC, hydroxylated GA1 and GA4 in preference to GA9 and GA20, while GA9 was the preferred substrate for the second enzyme (Mr 42,000). However, attempts to purify the enzyme activity to obtain N-terminal information for amino acid sequencing have proved impossible because of the low abundance of the enzyme in the plant tissues relative to other proteins and the co-purification of a contaminating lectin with the enzyme activity rendering N-terminal amino acid sequencing impossible.
The regulation of gibberellin deactivation has been examined in Pisum sativum (garden pea) using the sln (slender) mutation as reported in Ross et al (The Plant Journal 7 (3) 513-523 (1995)). The sln mutation blocks the deactivation of GA20 which is the precursor of the bioactive GA1. The results of these studies indicated that the sln gene may be a regulatory gene controlling the expression of two separate structural genes involved in GA deactivation, namely the oxidation of GA20 to GA29 by 2β-hydroxylation at C-2 followed by the further oxidation of the hydroxyl group to a ketone (GA29 to GA29 atabolite). The conversion of GA29 to GA29-cataholite in pea seeds was inhibited by prohexadione-calcium, an inhibitor of 2-oxoglutarate-dependent dioxygenases (Nakayama et al Plant Cell Physiol. 31 1183-1190 (1990)), indicating that the reaction was catalysed by an enzyme of this type. Although the slender (sin) mutation in peas was found to block both the conversion of GA20 to GA29 and of GA29 to GA29-catabolite in seeds, the inability of unlabeled GA20 to inhibit oxidation of radiolabelled GA29, and vice versa, indicated that the steps were catalysed by separate enzymes. Furthermore, in shoot tissues, the slender mutation inhibits the 2β-hydroxylation of GA20, but not the formation of GA29-catabolite. These observations lead to the theory that there were two separate enzymes involved in this metabolic pathway controlling the deactivation of GA in plants (Hedden, P. and Kamiya, Y., in Annu. Rev. Plant Physiol. Plant Mol. Biol. 48 431460 (1997)).